Escherichia coli: PhoP Locus
Introduction Escherichia coli is a gram-negative bacterium known as a model organism in microbiology due to its simple genome, competency as a host, rate of growth and ability to survive under both aerobic and anaerobic conditions. Since it resides within the gut of warm-blooded animals, E. coli serves as a critical model in studying disease of the gut and sepsis (1) The PhoP protein assists in the PhoPQ regulatory system that controls adaptation to Mg2+ limiting conditions in both the model organism, E. coli, and another bacteria, Salmonella typhimurium. While another protein, the PhoQ protein, acts as a sensor for extracytoplasmatic Mg2+ and Ca2+ (3), PhoP, who’s activity is governed by PhoQ, acts as a response regulator in modulating the necessary reaction to a particular stress (2). In E. coli, the PhoPQ system is not only an important component of the Mg2+ starvation response but is also important for antimicrobial peptide resistance (4,5). A molecular genetic analysis was done on the E. coli PhoP locus for comparison to the S. typhimurium ''homolog that’s known to encode a major virulence regulator necessary for intramacrophage survival along with phagocytic cationic peptide resistance(4). Genetic Analysis of PhoP Locus A mini-Mu replicon in vivo cloning procedure was used to prepare a library from the ''E. coli strain into the PhoP S. typhimurium strain. Kanamycin-resistant transductants were selected and screened for complementation of one of the phenotypes of S. typimurium, that of producing NSAP (following the procedure described by Kier et al. (6)). Restriction maps of the clones containing the PhoP homolog were then aligned with the physical map of the E. coli K-12 chromosome and a match to the desired region was found and verified via plaque hybridization. DNA sequence analysis was subsequently performed on the PhoP gene and it was determined to be transcribed in a counterclockwise direction in the E. coli chromosome and the deduced amino acid sequence of PhoP in E. coli was 93% identical and 98% similar to the S. typhimurium homolog. Lastly, a phenotype of a PhoP E. coli strain was determined via the construction of a mutant E. coli strain lacking the ability to produce NSAP (through an insertion in the PhoP open reading frame). The presence of a disrupted NSAP production function was confirmed with Southern hybridization analysis (4). The phenotypic differences between isogenic PhoP+ and PhoP E. coli were tested for via investigation of the effects of differing cationic peptide antibiotics that have been shown to have a detrimental effect on PhoP S. typhimurium (7). Differences in peptide susceptibility between PhoP mutants of E. coli ''and ''S. typhimurium were observed and may be attributed to differing sets of loci between the two species. Higher levels of resistance observed in the wild-type S. typhimurium strain compared to the wild-type E. coli strain suggests S. typhimurium ''contains a gene that allows it to withstand the microbicide effects of the peptides (4). Role of PhoP The role of PhoP as determined by the data presented in this genetic study includes involvement in response to stress such as those present during stationary phase, nutritional deprivation and exposure to metabolite toxins. Regulation of Phop in ''E. coli is controlled by regulatory genes such as the phosphate starvation-inducible psiG, psiH, psiI, psiJ, psiL ''and ''PsiO. It was observed that these genes don’t appear to be controlled by the PhoP homolog, PhoB, which regulates the expression of a multitude of phosphate starvation loci (8). The sequenced DNA region that precedes the PhoP coding region was found to have no resemblance to a heat shock promoter or the motifs found upstream of genes induced during stationary phase (9). Presence of a -10 region upstream of the PhoP gene was also located and suggests transcription by a α70 RNA polymerase. Overall, these deductions of the PhoP mutant in E. coli will allow for further research evaluation of the role of PhoP in response to an array of diverse environmental stresses (4). References #Hunter P. The paradox of model organisms. The use of model organisms in research will continue despite their shortcomings. EMBO. 9'(8):717-720 (2008). #Monsieurs P, De Keersmaecker S, Navarre WW, Bader MW, De Smet F, McClelland M, Fang FC, De Moor B, Vanderleyden J, Marchal K. Comparison of the PhoPQ Regulon in ''Escherichia coli and Salmonella typhimurium. J Mol Evol. '''60(4):462-474 (2005). #Vescovi EG, Ayala YM, Di Cera E, Groisman EA. Characterization of the bacterial sensory protein PhoQ. Evidence for distinct binding sites for Mg2+ and Ca2+. J Biol Chem. 272:1440-1443 (1997). #Groisman EA, Heffron F, Solomon F. Molecular Genetic Analysis of the Escherichia coli phoP Locus. Journal of Bacteriology. 174(2):486-491 (1992). #Groisman EA. The pleiotropic two-component regulatory system PhoP-PhoQ. Journal of Bacteriology. 183:1835-1842 (2001). #Kier LD, Weppelman RM, Ames BN. Regulation of two phosphatases and a cyclic phosphodiesterase of Salmonella typhimurium. Journal of Bacteriology. 130:420-428 (1977). #Groisman EA, Saier MH Jr.. Salmonella virulence: new clues to intramacrophage survival. Trends Biochem Sci. '15':30-33 (1990). # Wanner, BL. Phosphate regulation of gene expression in Escherichia coli, p. 1326-1333. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 2. 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